Condensed heterocyclic compounds, their production and use

ABSTRACT

A novel compound of the formula: ##STR1## wherein a ring A stands for an optionally substituted 5-membered ring; B stands for an optionally substituted divalent cyclic or chain group; either one of Q 1  and Q 2  stands for N and the other stands for N or CH; X stands for an amino group, hydroxyl group or mercapto group; Y stands for H, halogen atom or a group bonded through C, N, O or S; Z stands for a straight-chain divalent group having 2 to 5 atoms constituted of optionally substituted carbon atoms or constituted of optionally substituted carbon atoms and one optionally substituted hetero-atom; COOR 1  and COOR 2  independently stand for an optionally esterified carboxyl group; n denotes an integer of 2 to 6; and R 1  may be different in each of n repeating units, or their salts and it is useful as a therapeutic drug for tumor in mammals.

FIELD OF THE INVENTION

This invention relates to a novel condensed heterocyclic oligoglutamateuseful as an anti-tumor agent, its production and use.

BACKGROUND OF THE INVENTION

Folic acid and its related compounds are carriers of one-carbon (C1)units in a living body derived from formic acid or formaldehyde, actingas a coenzyme in various enzymatic reactions such as those inbiosynthesis of nucleic acid, in metabolism of amino acids and peptidesand in generation of methane. Particularly for metabolism or transferreaction of C1 units in biosynthesis of nucleic acid, i.e. the purinesynthetic pathway and the thymidine synthetic pathway, folic acid andits related compounds are essential. Usually, folic acid is efficientlystored intracellularly as oligoglutamates by glutamylation withfolylpolyglutamyl synthase after being transformed into tetrahydrofolicacid being an activated coenzyme by reduction in two steps. Thistetrahydrofolic acid and its oligoglutamates display biological activityas coenzyme in various enzymatic reactions in the state of combinationwith C1 units.

On the other hand, Amethopterin (methotrexate:MTX) and its relatedcompounds are known as drugs to inhibit the reduction from dihydrofolicacid into tetrahydrofolic acid by coupling strongly with the dominantenzyme (dihydrofolate reductase) in this reduction. These drugs havebeen developed as antitumor drugs because they may disturb the DNAsynthesis and consequently cause cell death, and are used clinically.Further, antifolates, having an action mechanism different frominhibition of dihydrofolate reductase, i.e. a tetrahydroaminopterinantitumor agent (5,10-dideaza-5,6,7,8-tetrahydroaminopterin : DDATHF),whose main action mechanism consists in inhibition of glycinamideribonucleotide transformylase concerned in the initial stage of purinebiosynthesis [Journal of Medicinal Chemistry, 28, 194 (1985)], and aquinazoline antitumor agent(2-desamino-2-methyl-10-propargyl-5,8-dideazafolate : DMPDDF), whosemain action mechanism consists in inhibition of thymidylate synthaseconcerned in transformation of deoxyuridin monophosphate todeoxythimidine monophosphate [British Journal of Cancer 58, 241 (1988)],and their oligoglutamates [Journal of Medicinal Chemistry, 29, 1754(1986) and 31. 181 (1988)] have been reported. However, theiroligoglutamates have as a condensed heterocyclic moiety a condensed ringformed with two 6-membered rings but do not have a condensed ring formedwith a 5-membered ring and 6-membered ring.

And, recently, it has been reported that, besides these antifolates andoligoglutamates having a condensed ring formed with a 6-membered ringand a 6-membered ring, compounds having a condensed ring formed with a6-membered ring and a 5-membered ring, i.e. a pyrrolo[2,3-d]pyrimidineskeleton, have also excellent anti-tumor activity. For example,EP-A-334636 describes a compound of the formula: ##STR2## wherein a ringA is a pyrrole or pyrroline ring, X is an amino group or a hydroxylgroup, Y is a hydrogen atom, an amino group or a hydroxyl group, R is ahydrogen atom, a fluorine atom, an alkyl group, an alkenyl group or analkynyl groups, --COOR¹ and --COOR² are independently carboxyl groupwhich may be esterified and n is an integer of 2 to 4, and R may bedifferent in each of the n repeating units, and salts thereof haveexcellent antitumor effects, and can be used as antitumor agents inmammals, EP-A-400562 describes a compound of the formula: ##STR3##wherein a ring A is a pyrrole ring which may be hydrogenated, X is anamino, hydroxyl or mercapto group, Y is a hydrogen atom or a hydroxylgroup, R¹, R², R⁴ and R⁵ may be the same or different and are each ahydrogen atom, a lower hydrocarbon radical or a chemical bond, Z is--O--, a group of the formula --S-- (O)n-in which n is an integer of 0to 2 or a group of the formula ##STR4## in which R³ is a hydrogen atom,a lower hydrocarbon radical optionally having substituent(s), a groupattached through --CO-- or --S(O)m (m is 1 or 2) or a chemical bond,--B-- is a divalent cyclic group optionally having substituent(s) of alower alkylene group optionally having substituent(s), --COOR⁶ and--COOR⁷ may be the same or different and are each a carboxyl group whichmay be esterified, and i and j are an integer of 0 to 3 provided thati+j=1 to 3, or its salt thereof, which is useful an antitumor agent,U.S. Pat. No. 4,996,206 describes a compound of the formula: ##STR5## inwhich

R¹ is --OH or --NH₂ ;

R³ is 1,4-phenylene or 1,3-phenylene unsubstituted or substituted withchloro, fluoro, methyl, methoxy, or trifluoromethyl: thienediyl orfuranediyl each unsubstituted or substituted with chloro, fluoro,methyl, methoxy, or trifluoromethyl: cyclohexanediyl: or alkanediyl:

R⁴ is hydrogen, methyl, or hydroxymethyl:

R⁵ is hydrogen or alkyl of 1 to 6 carbon atoms:

the configuration about the carbon atom designated* is S: and thepharmaceutically acceptable salts thereof, which is used asantineoplasmic agents, and U.S. Pat. No. 5,028,608 describes a compoundof the formula: ##STR6## in which

R¹ is --OH or --NH₂ :

R³ is thienediyl or furanediyl each unsubstituted or substituted withchloro, fluoro, methyl, methoxy, or trifluoromethyl; cyclohexanediyl; oralkanediyl;

R⁴ is hydrogen, methyl, or hydroxymethyl; the configuration about thecarbon atom designated* is S: and the pharmaceutically acceptable saltsthereof, which is used as antineoplasmic agents.

However, their compounds (A), (B), (C) and (D) having apyrrolo[2,3-d]pyrimidine skeleton are not in the form of oligoglutamatesat their side chain.

What is now specifically desired in the field of cancer therapy is thecreation of drugs which have toxicities highly specific to cancer cellsbased on the action mechanism having excellent therapeutic effects. TheMTX whose principal action mechanism consists in inhibition ofdihydrofolate reductase is clinically used widely, though thetherapeutic effect is still unsatisfactory because is has relativelystrong toxicity with little effect on solid cancer.

SUMMARY OF THE INVENTION

As the result of the inventors' diligent research under thecircumstances described above, they have found that noveloligoglutamates having a ring condensed heterocyclic formed with certain6-membered ring and 5-membered ring are stored in cells efficiently, andperform excellent anti-tumor action while showing highly specifictoxicities to various cells, especially to solid tumors, by inhibitingone or more pathways of biosynthesis of nucleic acid, with which folicacid and its related compounds are concerned and have an excellentsolubility to water.

DETAILED DESCRIPTION OF THE INVENTION

Namely, this invention provides (1) compounds of the formula (I):##STR7## wherein a ring A stands for an optionally substituted5-membered ring; B stands for an optionally substituted divalent cyclicor chain group; either one of Q¹ and Q² stands for N and the otherstands for N or CH; X stands for an amino group, hydroxyl group ormercapto group; Y stands for hydrogen atom, a halogen atom or a groupbonded through carbon, nitrogen, oxygen or sulfur atom; Z stands for astraight-chain divalent group having 2 to 5 atoms constituted ofoptionally substituted carbon atoms or constituted of optionallysubstituted carbon atoms and one optionally substituted hetero atom;COOR¹ and COOR² independently stand for an optionally esterifiedcarboxyl group; n denotes an integer of 2 to 6; and R¹ may be differentin each of n repeating units, or their salts,

(2) a method of producing a compound of the formula (I) or a saltthereof, which is characterized by allowing a compound of the formula(II): ##STR8## wherein the symbols are of the same meaning as defined inthe formula (I), or a reactive derivative thereof at the carboxyl groupto react with a compound of the formula (III): ##STR9## wherein thesymbols are of the same meaning as defined in the formula (I), and

(3) an anti-tumor composition comprising a compound of the formula (I)or a salt thereof.

Among the compounds of the formula (I), especially preferable ones arethose represented by the formula (IV): ##STR10## wherein a ring A standsfor an optionally hydrogenated pyrrole ring, R stands for hydrogen or alower alkyl group, m denotes an integer of 2 to 4, R may be different ineach of m repeating units, and the other symbols are of the same meaningas defined in the formula (I).

When X in the formulae described above is a hydroxyl group or mercaptogroup, each of the compounds (I), (II) and (IV) may exist as anequilibrium mixture of the respective tautomers. The following partialstructural formulae show the sites of the structure which are subject totautomerism, with the equilibrium relationship between the tautomers.##STR11##

For the convenience of description, hydroxyl forms or mercapto forms andthe corresponding names are described throughout this specification, butthe corresponding oxo forms or thio forms are included as well.

There may be two or more asymmetric centers in the compounds (I) orsalts thereof of this invention, and the absolute configuration of theasymmetric centers may be S, R or RS mixed form, except that theabsolute configuration at the asymmetric carbon atom in the side chainderived from glutamic acid is always S(L). Therefore, the compounds (I)or salts thereof may have two or more diastereomers which, whennecessary, can easily be separated by a conventional method forseparation and purification. All of the diastereomers which can beseparated thus are included in this invention.

Examples of the 5-membered cyclic groups shown by the ring A in theformula (I) described above include cyclic groups composed of carbonatoms, or carbon atoms and one hetero atom (nitrogen atom, oxygen atomor sulfur atom), and these cyclic groups may be substituted.

Examples of the ring of these cyclic groups include cyclopentadiene,cyclopentene, furan, dihydrofuran, thiophene, dihydrothiophene,thiophen-1-oxide, dihydrothiophen-1-oxide, thiophen-1,1-dioxide,dihydrothiophen-1,1-dioxide, pyrrole, pyrroline, N-substituted pyrroleand N-substituted pyrroline.

These cyclic groups may have 1 or 2 substituents at any possibleposition, and examples of said substituents include a C₁₋₃ alkyl group(e.g. methyl, ethyl, propyl, iso-propyl group), C₂₋₃ alkenyl group (e.g.vinyl, 1-methylvinyl, 1-propenyl, allyl, allenyl group), C₂₋₃ alkynylgroup (e.g. ethynyl, 1-propynyl, propargyl group), C₃₋₆ cycloalkyl group(e.g. cyclopropyl group), halogen atom, (e.g. fluorine, chlorine,bromine, iodine), C₁₋₄ alkanoyl group (e.g. formyl, acetyl, propionyl,butyryl, isobutyryl group), benzoyl group, substituted benzoyl group,preferably a benzoyl group substituted with 1 to 3 substituents selectedfrom a halogen atom and a C₁₋₄ alkoxy group (e.g. p-chlorobenzoyl,p-methoxybenzoyl, 3,4,5-trimethoxybenzoyl group), cyano group, carboxylgroup, carbamoyl group, nitro group, hydroxyl group, hydroxy-C₁₋₃ alkylgroup (e.g. hydroxymethyl group, hydroxyethyl group), C₁₋₃ alkoxy-C₁₋₃alkyl group (e.g.methoxymethyl group, methoxyethyl group, ethoxyethylgroup), C₁₋₃ alkoxy group (e.g. methoxy, ethoxy, propoxy group),mercapto group, C₁₋₃ alkylthio group (e.g. methylthio, ethylthio,propylthio group), amino group, C₁₋₄ substituted amino group, preferablyan amino group substituted with one or two C₁₋₄ alkyl groups (e.g.methylamino, ethylamino, dimethylamino, diethylamino group), and C₁₋₂alkanoylamino group (e.g. formamido, acelamido group).

Examples of the N-substituents at the N-substituted pyrrole andN-substituted pyrroline rings include, the above-mentioned C₁₋₃ alkylgroup, C₂₋₃ alkenyl group, C₃₋₆ cycloalkyl group (e.g. cyclopropylgroup), C₁₋₄ alkanoyl group, benzoyl group, substituted benzoyl group,hydroxyethyl group, methoxyethyl group and ethoxyethyl group, andbesides a phenyl group, a substituted phenyl group, preferably a phenylgroup substituted with 1 to 3 substituents selected from a halogen atomand a C₁₋₄ alkoxy group (e.g. p-chlorophenyl, p-methoxyphenyl,3,4,5-trimethoxyphenyl group), a benzyl group or substituted benzylgroup, preferably a benzyl group substituted with 1 to 3 substituentsselected from a halogen atom, a C₁₋₄ alkoxy group and phenyl (e.g.p-chlorobenzyl, p-methoxybenzyl, diphenylmethyl group). The ring A canbe bonded to Z at any possible position, and, in the case of the ring Abeing a N-substituted pyrrole or N-substituted pyrroline ring, thebondage may occur at the N-substituted portion.

Preferable cyclic groups in an optionally substituted divalent cyclicgroup shown by B include a 5- or 6-membered divalent cyclic groupoptionally containing a hetero atom (e.g. N, O, S), having bonding handsat positions which are not adjacent to each other in the ring. Examplesof the 5-membered divalent cyclic group shown by B include 1,3- or3,5-cyclopentadien-1,3-ylene, cyclopenten-(1,3-, 1,4- or 3,5-)ylene,cyclopentan-1,3-ylene, thiophen-(2,4-, 2,5- or 3,4-)ylene, pyrrol-(1,3-,2,4-, 2,5- or 3,4-)ylene, thiazol-(2,4- or 2,5-)ylene, imidazol-(1,4-,2,4- or 2,5-)ylene, thiadiazol-2,5-ylene or their partially orcompletely reduced derivatives (e.g. (2,3- or 4,5-)dihydropyrrol-(2,4-,2,5- or 3,4-)ylene, 2,3,4,5-tetrahydropyrrol-(2,4-, 2,5- or 3,4-)ylene).

And, examples of the 6-membered divalent cyclic group includephenyl-(1,3- or 1,4)ylene, cyclohexan-(1,3- or 1,4-)ylene,cyclohexen-(1,3-, 1,4-, 1,5-, 3,5- or 3,6-)ylene,1,3-cyclohexadien-(1,3-, 1,4-, 1,5-, 2,4-, 2,5- or 2,6-)ylene,1,4-cyclohexadien-(1,3-, 1,4- or 1,5-)ylene, pyridin-(2,4-, 2,5-, 2,6-or 3,6-)ylene, pyran-(2,4-, 2,5-, 2,6-, 3,5-, 3,6- or 4,6-)ylene,pyrazin-(2,5- or 2,6-)ylene, pyrimidin-(2,4- or 2,5-)ylene,pyridazin-3,5-ylene or their partially or completely reduced derivatives(e.g. piperidin-(2,4-, 2,5-, 2,6- or 3,6-)ylene, piperazin-(2,5- or2,6-)ylene). Among them, phenyl-1,4-ylene and thiophen-2,5-ylene, etc.are especially preferable.

Preferable chain groups in an optionally substituted divalent chaingroup shown by B include a divalent C₂₋₄ chain-like hydrocarbon groupssuch as ethylene, ethenylene, ethynylene, trimethylene, propenylene,propynylene, propadienylene, tetramethylene, butenylene, butynylene orbutanedienylene, etc.

Divalent cyclic or chain groups shown by B may have 1 or 2 substituentsat any possible position. Examples of such substituents include C₁₋₃alkyl groups (e.g. methyl, ethyl, propyl, isopropyl group), C₂₋₃ alkenylgroups (e.g. vinyl, 1-propenyl, allyl, allenyl group), C₂₋₃ alkynylgroups (ethynyl, 1-propynyl, propargyl group), C₃₋₆ cycloalkyl groups(e.g. cyclopropyl), halogen (e.g. chlorine, bromine, fluorine, iodine),hydroxyl, C₁₋₄ alkoxy groups (e.g. methoxy), di-C₁₋₄ alkylamino groups(e.g. dimethylamino), halogeno-C₁₋₄ alkyl groups (e.g. trifluoromethyl),oxo, C₁₋₃ acyl groups (e.g. formyl), C₁₋₃ alkoxy-C₁₋₃ alkyl groups (e.g.methoxymethyl, 2-ethoxyethyl), etc.

Y can be a cyano group, carboxyl group, carbamoyl group, nitro group,hydroxyl group, amino group or a lower hydrocarbon group, for example,C₁₋₃ alkyl group (e.g. methyl, ethyl, propyl, iso-propyl group), C₂₋₃alkenyl group (e.g. vinyl, 1-methylvinyl, 1-propenyl, allyl, allenylgroup), C₂₋₃ alkynyl group ( e.g. ethynyl, 1-propinyl, propargyl group)and C₃₋₆ cycloalkyl group (e.g. cyclopropyl group), etc., aryl group,preferably C₆₋₁₀ aryl group such as phenyl group, naphthyl group; 5- or6-membered heterocyclic group containing 1 to 3 hetero atoms selectedfrom nitrogen, oxygen and sulfur atom such as pyrrolyl, imidazolyl,pyrazolyl, thienyl, furyl, thiazolyl, thiadiazolyl, oxazolyl,oxadiazolyl, pyridyl, pyranyl, pyrazinyl, pyrimidinyl, pyridazinyl ortheir partially or completely reduced derivatives (e.g.2,3,4,5-tetrahydropyrrolyl, (1,2,3,4-, 1,2,5,6- or3,4,5,6-)tetrahydropyridyl), dioxoranyl, piperidino, morpholino,N-methylpiperazinyl, N-ethylpiperazinyl, dioxanyl, etc.

When Y is a lower hydrocarbon group, aryl group or a 5- or 6-memberedheterocyclic group, it may have 1 or 2 substituents. Examples of suchsubstituents include a C₁₋₃ alkyl group (e.g. methyl, ethyl propyl,isopropyl group), C₂₋₃ alkenyl group (e.g. vinyl, 1-methylvinyl,1-propenyl, allyl, allenyl group), C₂₋₃ alkynyl group (ethynyl,1-propinyl, propargyl group) or C₃₋₆ cycloalkyl group (e.g. cyclopropylgroup), and, besides, halogen (e.g. fluorine), hydroxyl, oxo, a C₁₋₄alkoxy group (e.g. methoxy), di-C₁₋₄ alkylamino group (e.g.dimethylamino, diethylamino), halogeno-C₁₋₄ trifluoromethyl), C₁₋₃ acyl(e.g. formyl), hydroxy-C₁₋₄ alkyl (e.g. hydroxymethyl, 2-hydroxyethyl),C₁₋₄ alkoxy-C₁₋₄ alkyl e.g. methoxymethyl, 2-ethoxyethyl), etc.

Examples of a halogen atom shown by Y include fluorine, chlorine,bromine or iodine. Y may be an alkoxy group, alkylthio group,alkanoylamino group or alkanoyloxy group, and, as the alkyl portion ofthese groups, mention is made of those optionally substituted asexemplified in the case where Y is a lower hydrocarbon group. Y may bean aryloxy group, arylthio group, aroylamino group or aroyloxy group,and, as the aryl portion of these groups, mention is made of thoseoptionally substituted as exemplified in the case where Y is an arylgroup. Further, Y may be a heterocyclicoxy group, heterocyclic-thiogroup, heterocyclic-carbonylamino group or heterocyclic-carbonyloxygroup, and, as the heterocyclic portion of these groups, mention is madeof groups optionally substituted as exemplified in the case where Y is a5- to 6-membered heterocyclic group. Y may be a substituted amino groupsuch as a mono-substituted or di-substituted amino group, and, as thesubstituent of the substituted amino group, mention is made of theabove-mentioned lower hydrocarbon groups, aryl group and 5- to6-membered heterocyclic groups.

In the straight-chain divalent group having 2 to 5 atoms shown by Z,which is constituted of optionally substituted carbon atoms orconstituted of optionally substituted carbon atoms and one optionallysubstituted hetero-atom (nitrogen atom, oxygen atom or sulfur atom),examples of the group constituted of carbon atoms include C₂₋₅ alkylenegroups such as ethylene, trimethylene, tetramethylene, pentamethylene,etc., C₂₋₅ alkenylene qroups such as vinylene, propenylene, 1- or2-butenylene, butadienylene, 1- or 2-pentenylene, 1,3- or1,4-pentadienylene, etc. and C₂₋₅ alkynylene groups such as ethynylene,1- or 2-propynylene, 1- or 2-butynylene, 1-, 2- or 3-pentynylene, etc.

As the group constituted of optionally substituted carbon atoms and oneoptionally substituted hetero-atom (nitrogen atom, oxygen atom or sulfuratom), mention is made of a group shown by --Z¹ --Z² --Z³ -- wherein Z¹and Z³ independently stand for a bonding hand or an optionallysubstituted divalent lower hydrocarbon group, and Z² stands for --O--, agroup shown by the formula: --S(O)p- wherein p denotes an integer of 0to 2, or a group of the formula: --NR³ -- wherein R³ stands for hydrogenatom or an optionally substituted lower hydrocarbon group. Examples ofthe divalent lower hydrocarbon group shown by Z¹ and Z³ include C₁₋₄alkylene groups such as methylene, ethylene, trimethylene,tetramethylene, etc., C₂₋₄ alkenylene groups such as vinylene,propenylene, 1- or 2-butenylene, butadienylene, etc., and C₂₋₄alkynylene groups such as ethynylene, 1- or 2-propynylene, 1- or2-butynylene, etc.

Examples of the lower hydrocarbon group shown by R³ include C₁₋₃ alkylgroups (e.g. methyl, ethyl, propyl, iso-propyl group), C₂₋₃ alkenylgroups (e.g. vinyl, 1-methylvinyl, 1-propenyl, ally, allenyl group),C₂₋₃ alkynyl groups (e.g. ethynyl, 1-propynyl, propargyl group) and C₃₋₆cycloalkyl (e.g. cyclopropyl group), etc. The straight-chain divalentgroup having 2 to 5 atoms constituted of carbon atoms of Z, the divalentlower hydrocarbon group of Z¹ and Z³ and the lower hydrocarbon group ofR³ may have one or two substituents, and examples of such substituentsinclude a C₁₋₃ alkyl group (e.g. methyl, ethyl propyl, iso-propylgroup), C₂₋₃ alkenyl group (e.g. vinyl, 1-methylvinyl, 1-propenyl,allyl, allenyl group), C₂₋₃ alkynyl group (e.g. ethynyl, 1-propinyl,propargyl group), C₃₋₆ cycloalkyl group (e.g. cyclopropyl group), and,besides, a halogen (e.g. fluorine), hydroxyl, oxo, C₁₋₃ alkoxy group(e.g. methoxy), di-C₁₋₃ alkyl-amino group (e.g. dimethylamino,diethylamino), halogeno-C₁₋₃ alkyl group (e.g. trifluoromethyl), C₁₋₃acyl (e.g. formyl), hydroxy-C₁₋₃ alkyl group (e.g. hydroxymethyl,2-hydroxyethyl), C₁₋₃ alkoxy-C₁₋₃ alkyl group (e.g. methoxymethyl,2-ethoxyethyl), etc.

In a preferable compound (IV) of the present invention, as an optionallyhydrogenated pyrrole ring shown by A, mention is made of a pyrrole ringand pyrroline ring, and, as a lower alkyl group shown by R, mention ismade of C₁₋₃ alkyl groups (e.g. methyl, ethyl, propyl, iso-propylgroup).

Examples of optionally esterified carboxyl groups shown by --COOR¹ and--COOR² include carboxyl groups optionally esterified with, amongothers, a C₁₋₅ lower alkyl group (e.g. methyl, ethyl, propyl,iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl,iso-pentyl, sec-pentyl, neo-pentyl, tert-pentyl group, etc.), anoptionally substituted benzyl group, preferably benzyl group which maybe substituted with nitro or a C₁₋₄ alkoxy group (e.g. benzyl,nitrobenzyl, methoxybenzyl group, etc.) or an optionally substitutedphenyl group, preferably phenyl group which may be substituted withnitro or a C₁₋₄ alkoxy group (e.g. phenyl, nitrophenyl, methoxyphenylgroup, etc.).

m denotes an integer of 2 to 4, preferably 3, n denotes an integer of 2to 6, preferably 2 to 4, respectively, and R¹ may be different in eachof n repeating units, and R may be different in each of m repeatingunits, respectively.

Typical examples of the compounds (I) of the present invention include

1)[N-[4-[2-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

2)[N-[4-[3-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

3)[N-[5-[2-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

4)[N-[5-[3-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)propyl]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

5)[N-[4-[N-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)methylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

6)[N-[4-[N-2-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)ethylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

7)[N-[4-[N-[(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)methyl]-N-methylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

8)[N-[4-[N-[2-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

9)[N-[4-[N-[(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)methyl]-N-propargylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

10)[N-[4-[N-[2-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

11)[N-[5-[N-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)methylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

12)[N-[5-[N-2-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)ethylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

13)[N-[5-[N-[(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)methyl]-N-methylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

14)[N-[5-[N-[2-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

15)[N-[5-[N-[(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)methyl]-N-propargylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

16)[N-[5-[N-[2-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

17)[N-[4-[2-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-6-yl)ethyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

18)[N-[4-[3-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-6-yl)propyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

19)[N-[5-[2-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-6-yl)ethyl]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

20)[N-[5-[3-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-6-yl)propyl]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

21)[N-[4-[N-[(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-6-yl)methyl]-N-methylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

22)[N-[4-[N-2-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-6-yl)ethyl]-N-methylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

23)[N-[5-[N-[(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-6-yl)methyl]-N-methylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

24)[N-[5-[N-[2-(2-amino-4-hydroxy-7-methylpyrrolo[2,3-d]pyrimidin-6-yl)ethyl]-N-methylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

25)[N-[4-[2-(2-amino-4-hydroxycyclopentapyrimidin-5-yl)ethyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

26)[N-[4-[3-(2-amino-4-hydroxycyclopentapyrimidin-5-yl)propyl]benzoyl]-.gamma.-L-glutamyl]-L-glutamicacid,

27)[N-[4-[2-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-.gamma.-L-glutamyl]-L-glutamicacid,

28)[N-[4-[2-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

29)[N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

30)[N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-1-methylpropyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

31)[N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)-1-methylpropyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

32)[N-[4-[2-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-3-chlorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

33)[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-3-chlorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

34)[N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]-3-chlorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

35)[N-[5-[2-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-thenoyl]-.gamma.-L-glutamyl]-L-glutamicacid,

36)[N-[5-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

37)[N-[4-[N-2-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

38)[N-[4-[N-2-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

39)[N-[4-[N-[2-(2,4-diamino-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

40)[N-[4-[N-[2-(2,4-diamino-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-(tert-butoxycarbonyl)amino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

41)[N-[4-[2-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl]benzoyl]-.gamma.-L-glutamicacid,

42)[N-[4-[2-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

43)[N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)propyl]benzoyl]-.gamma.-L-glutamyl]-L-glutamicacid,

44)[N-[4-[3-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-6-yl)propyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

45)[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

46)[N-[4-[2-(2-amino-4-hydroxy-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

47)[N-[4-[3-(2-amino-4-hydroxy-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

48)[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

49)[N-[4-[2-(2-amino-4-hydroxy-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

50)[N-[4-[3-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-6-yl)propyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

51)[N-[4-[3-(2-amino-4-hydroxy-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-6-yl)propyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

52)[N-[4-[2-(2,4-diamino-furo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-γ-glutamyl]-L-glutamicacid,

53)[N-[4-[2-(2-amino-4-hydroxyfuro[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-.gamma.-L-glutamyl]-L-glutamicacid,

54)[N-[4-[3-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

55)[N-[4-[3-(2-amino-4-hydroxyfuro[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-.gamma.-L-glutamyl]-L-glutamicacid,

56)[N-[4-[2-(2,4-diaminothieno[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

57)[N-[4-[2-(2-amino-4-hydroxythieno[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-.gamma.-L-glutamyl]-L-glutamicacid,

58)[N-[5-[2-(2,4-diaminothieno[2,3-d]pyrimidin-5-yl)ethyl]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

59)[N-[5-[2-(2-amino-4-hydroxythieno[2,3-d]pyrimidin-5-yl)ethyl]-2-thenoyl]-.gamma.-L-glutamyl]-L-glutamicacid,

60)[N-[4-[3-(2,4-diaminothieno[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

61)[N-[4-[3-(2-amino-4-hydroxythieno[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-.gamma.-L-glutamyl]-L-glutamicacid,

62) [N-[5-[3-(2,4-diaminothieno[2,3-d]pyrimidin-5-yl)propyl]-2-thenoyl]-γ-L-glutamyl]-L-glutamic acid,

63)[N-[5-[3-(2-amino-4-hydroxythieno[2,3-d]pyrimidin-5-yl)propyl]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

64)[N-[4-[2-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethylthio]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

65)[N-[4-[3-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

66)[N-[4-[N-[2-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]aminobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

67)[N-[5-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]-2-thenoyl]-.gamma.-L-glutamyl]-L-glutamicacid,

68)[N-[4-[N-2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

69)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

70)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

71)[N-[5-[N-2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

72)[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

73)[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl-N-propargylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

74)[N-[4-[N-(2-amino-4-hydroxythieno[2,3-d]pyrimidin-5-yl)methylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

75)[N-[4-[N-[(2-amino-4-hydroxythieno[2,3-d]pyrimidin-5-yl)methyl]-N-methylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

76)[N-[4-[N-[(2-amino-4-hydroxythieno[2,3-d]pyrimidin-5-yl)methyl]-N-propargylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid,

77)[N-[5-[N-(2-amino-4-hydroxythieno[2,3-d]pyrimidin-5-yl)methylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

78)[N-[5-[N-[(2-amino-4-hydroxythieno[2,3-d]pyrimidin-5-yl)methyl]-N-methylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

79)[N-[5-[N-[(2-amino-4-hydroxythieno[2,3-d]pyrimidin-5-yl)methyl]-N-propargylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid.

80)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-2-fluorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

81)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-2fluorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

82)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]-2-fluorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

83)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]-2-fluorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

84)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]-2-chlorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

85)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]-2-chlorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

86)[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]thiazol-2-ylcarbonyl]-γ-L-glutamyl]-L-glutamicacid,

87)[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]thiazol-2-ylcarbonyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

88)[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]pyridin-2-ylcarbonyl]-γ-L-glutamyl]-L-glutamicacid,

89)[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]pyridin-2-ylcarbonyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

90)[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]thiazol-2-ylcarbonyl]-γ-L-glutamyl]-L-glutamicacid,

91)[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]thiazol-2-ylcarbonyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

92)[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]pyridin-2-ylcarbonyl]-γ-L-glutamyl]-L-glutamicacid,

93)[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]pyridin-2-ylcarbonyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

94)[N-[4-[2-(4-hydroxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-chlorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

95)[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-chlorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

96)[N-[4-[2-(4-hydroxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-fluorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

97)[N-[4-[2-(4-hydroxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-fluorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

98)[N-[4-[2-(4-hydroxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-3-fluorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

99)[N-[4-[2-(4-hydroxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-3-chlorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

100)[N-[5-[2-(4-hydroxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid,

101)[N-[5-[2-(4-hydroxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-thenoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

102)[N-[5-[2-(4-hydroxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]thiazol-2-ylcarbonyl]-γ-L-glutamyl]-L-glutamicacid,

103)[N-[5-[2-(4-hydroxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]thiazol-2-ylcarbonyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

104)[N-[5-[2-(4-hydroxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]pyridin-2-ylcarbonyl]-γ-L-glutamyl]-L-glutamicacid,

105)[N-[5-[2-(4-hydroxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]pyridin-2-ylcarbonyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

106)[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-chlorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

107)[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-chlorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

108)[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-fluorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

109)[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-fluorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

110)[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-3-fluorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

111)[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-3-fluorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

112)[N-[5-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]thiazol-2-ylcarbonyl]-γ-L-glutamyl]-L-glutamicacid,

113)[N-[5-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]thiazol-2-ylcarbonyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

114)[N-[5-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]pyridin-2-ylcarbonyl]-γ-L-glutamyl]-L-glutamicacid,

115)[N-[5-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]pyridin-2-ylcarbonyl]-γ-L-glutamyl)-γ-L-glutamyl-L-glutamicacid,

116)[N-[4-[2-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-.gamma.-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

117)[N-[4-[2-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-3-chlorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-qlutamicacid,

118)[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-3-chlorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

119)[N-[5-[2-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-thenoyl]-.gamma.-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

120)[N-[5-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-2-thenoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

121)[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

122)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]benzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

123)[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-2-thenoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

124)[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]-2-thenoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

125)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-3-fluorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

126)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-3-fluorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

127)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]-3-fluorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

128)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]-3-fluorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

129)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-2-chlorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

130)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-3-chlorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

131)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]-3-chlorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

132)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]-3-chlorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid,

133)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-3-chlorobenzoyl]-γ-L-glutamyl]-L-glutamicacid,

134)[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-3-chlorobenzoyl]-γ-L-glutamyl]-γ-L-glutamyl-L-glutamicacid, and all the triglutamate corresponding to the above examples ofdiglutamates.

In the following, the method for production of the compounds (I) of thisinvention is explained.

The compounds (I) or salts thereof can be obtained by acylation ofoligoglutamic acid derivatives shown by the formula (III) withcarboxylic acids shown by the formula (II) or its reactive derivativesat the carboxyl group. The acylation may be performed by, for example,acylation of the compound (III) with the compound (II) or a reactivederivative thereof in the presence of carbodiimide, diphenylphosphorylazide or diethyl cyanophosphonate. Generally, about 1 to 20 moleequivalents, preferably about 1 to 5 mole equivalents of the compound(III) are used relative to the compound (II) or its reactive derivative.Generally, about 1 to 25 mole equivalents, preferably about 1 to 5 moleequivalents of carbodiimide, diphenylphosphoryl azide or diethylcyanophosphonate are used relative to the compound (II) or its reactivederivative. While, as the carbodiimide, dicyclohexylcarbodiimide ispreferable for practical use, other carbodiimides such asdiphenylcarbodiimide, di-o-tolylcarbodiimide, di-p-tolylcarbodiimide,di-tert-butylcarbodiimide,1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide,1-cyclohexyl-3-(4-diethylaminocyclohexyl)carbodiimide,1-ethyl-3-(2-diethylaminopropyl)carbodiimide and1-ethyl-3-(3-diethylaminopropyl)carbodiimide may be used.

The acylation is preferably performed in the presence of a suitablesolvent, for example, water, alcohols (e.g. methanol, ethanol), ethers(e.g. dimethyl ether, diethyl ether, tetrahydrofuran, dioxane,monoglyme, diglyme), nitriles (e.g. acetonitrile), esters (e.g. ethylacetate), halogenated hydrocarbon (e.g. dichloromethane, chloroform,carbon tetrachloride), aromatic hydrocarbon (e.g. benzene, toluene,xylene), acetone, nitromethane, pyridine, dimethylsulfoxide,dimethylformamide, hexamethylphosphoramide, sulfolane or a suitablemixture of these solvents. This reaction is allowed to proceed usuallyat a pH ranging from about 2 to 14, preferably from about 6 to 9, at atemperature ranging from -10° C. to around the boiling point of thesolvent then used (up to about 100° C.), preferably at a temperatureranging from about 0° to 50° C., for about 1 to 100 hours, preferablyfor about 2 to 48 hours. The pH of the reaction mixture is adjusted,upon necessity, by the addition of an acid (e.g. hydrochloric acid,sulfuric acid, phosphoric acid, nitric acid, acetic acid), a base (e.g.sodium methylate, sodium ethylate, sodium hydroxide, potassiumhydroxide, barium hydroxide, lithium hydroxide, sodium carbonate,potassium carbonate, barium carbonate, calcium carbonate, sodiumhydrogencarbonate, trimethylamine, triethylamine, triethanolamine,pyridine) or a buffer solution (e.g. phosphate buffer, borate buffer,acetate buffer), etc.

The reaction can be allowed to proceed more advantageously by using acatalyst capable of promoting acylation. Examples of such catalystsinclude base catalysts and acid catalysts. The base catalysts includetertiary amines (e.g. aliphatic tertiary amines such as triethylamine;aromatic tertiary amines such as pyridine, α-, β- or γ-picoline,2,6-lutidine, 4-dimethylaminopyridine, 4-(1-pyrrolidinyl)pyridine,dimethylaniline and diethylaniline), and such acid catalysts includeLewis acids [e.g. anhydrous zinc chloride, anhydrous aluminum chloride(AlCl₃), anhydrous ferric chloride, titanium tetrachloride (TiCl₄), tintetrachloride (SnCl₄), antimony pentachloride, cobalt chloride, cupricchloride, boron trifluoride etherate, etc.]. Among the catalystsdescribed above, 4-dimethylaminopyridine or 4-(1-pyrrolidinyl)pyridineis preferable in many cases. The suitable amount of the catalyst is suchthat is enough to promote the acylation, being generally about 0.01 to10 mole equivalents, preferably about 0.1 to 1 mole equivalent relativeto the compound (II) or its reactive derivative. The reactivederivatives of the compound (II) at the carboxyl group, which are to beemployed for the acylation, include acid halides (e.g. fluoride,chloride, bromide, iodide), acid anhydrides (e.g. iodoacetic acidanhydride, isobutyric acid anhydride), mixed acid anhydrides withmonoalkylcarbonic acid esters (e.g. monomethylcarbonic acid ester,monoethylcarbonic acid ester, monopropylcarbonic acid ester,mono-iso-propylcarbonic acid ester, monobutylcarbonic acid ester,mono-isobutylcarbonic acid ester, mono-sec-butylcarbonic acid ester,mono-tert-butylcarbonic acid ester), active esters (e.g. cyanomethylester, carboethoxymethyl ester, methoxymethyl ester, phenyl ester,o-nitrophenyl ester, p-nitrophenyl ester, p-carbomethoxyphenyl ester,p-cyanophenyl ester, thiophenyl ester), acid azides, mixed acidanhydrides with phosphoric acid diesters (e.g. dimethyl phosphate,diethyl phosphate, dibenzylphosphate, diphenylphosphate), and mixed acidanhydrides with phosphorous acid diesters (e.g. dimethyl phosphite,diethyl phosphite, dibenzyl phosphite, diphenyl phosphite), of thecarboxylic acid (II). For acylation with such a reactive derivative, thesolvent, the catalyst and the reaction temperature are the same as foracylation in the presence of the carbodiimide, diphenylphosphoryl azideor diethyl cyanophosphonate described above.

For production of the compound (I-1) in which --COOR¹ and --COOR² in theformula (I) are carboxyl groups, or a salt thereof, it is preferablethat the compound, in which --COOR¹ and --COOR² in the formula of thecompound (III) are esterfied carboxyl groups, is allowed to react withthe compound (II) or its reactive derivative at the carboxyl group asmentioned above, followed by deesterification by per se knowndegradation or catalytic reduction.

Such degradation can be performed by hydrolysis under basic conditions(method A), hydrolysis under acid conditions (method B-1) or hydrolysisunder acid and non-aqueous conditions (method B-2). Bases used in themethod A include metal alkoxides such as sodium methoxide, sodiumethoxide, sodium butoxide and potassium butoxide, metal hydroxides suchas sodium hydroxide, potassium hydroxide, lithium hydroxide, and bariumhydroxide, and amines such as ammonia, triethylamine and pyridine. Acidsused in the method B-1 include mineral acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid, andorganic acids such as trifluoroacetic acid, trichloroacetic acid,methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid andcamphorsulfonic acid. Acids usable in the method B-2 include mineralacids such as hydrogen chloride, hydrogen bromide, perchloric acid,sulfuric acid, nitric acid and phosphoric acid, organic acids such astrifluoroacetic acid, trichloroacetic acid, methanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid and camphorsulfonic acid,and Lewis acids such as anhydrous zinc chloride, anhydrous aluminumchloride (AlCl₃), anhydrous ferric chloride, titanium tetrachloride(TiCl₄), tin tetrachloride (SnCl₄), antimony pentachloride, cobaltchloride, cupric chloride and boron trifluoride etherate. Degradation isperformed in a suitable solvent at a temperature ranging from 0° C. tothe boiling point of the solvent, preferably at 10° to 80° C., for 30minutes to two days. The solvent usable for the reaction in the method Aor the method B-1 may be water, methanol, ethanol, propanol, butanol,ethylene glycol, methoxyethanol, ethoxyethanol, tetrahydrofuran,dioxane, monoglyme, diglyme, pyridine, dimethylformamide, dimethylsulfoxide, sulfolane, or a mixture of them; the solvents usable for thereaction in the method B-2 may be ethyl acetate, dimethyl ether, diethylether, tetrahydrofuran, dioxane, monoglyme, diglyme, dichloromethane,chloroform, carbon tetrachloride, acetonitrile, benzene, toluene,xylene, nitromethane, pyridine or a suitable mixture of them.

The catalytic reduction (method C) is performed in a suitable solvent ata temperature ranging from about -40° C. to the boiling point of thesolvent used, preferably at about 0° to 50° C. The solvents usableinclude water, alcohols (e.g. methanol, ethanol, propanol, iso-propanol,butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, ethylene glycol,methoxyethanol, ethoxyethanol), acetic acid esters (e.g. methyl acetate,ethyl acetate), ethers (e.g. dimethyl ether, diethyl ether,tetrahydrofuran, dioxane, monoglyme, diglyme), aromatic hydrocarbons(e.g. benzene, toluene, xylene), pyridine, dimethylformamide and asuitable mixture of them. Examples of catalysts for the catalyticreaction include palladium, platinum, rhodium and Raney's nickel.Addition of a trace amount of acetic acid, trifluoroacetic acid,hydrochloric acid or sulfuric acid sometimes serves to allow thereaction to proceed advantageously.

The method for production of the compound (I-1) or a salt thereof isselected according to the nature of --COOR¹ and --COOR² in the startingcompound (III); when --COOR¹ and --COOR² in the compound (III) arecarboxyl groups esterified with a methyl, ethyl, propyl, butyl,sec-butyl, phenyl or substituted phenyl group, the method A or themethod B-1 is applied advantageously; when --COOR¹ and COOR² arecarboxyl groups esterified with an iso-propyl or tert-butyl group, themethod B-2 is applied advantageously; and when --COOR¹ and --COOR² arecarboxyl groups esterified with a benzyl or a substituted benzyl group,the method B-1 or the method C is applied advantageously. When --COOR¹and --COOR² are different from each other, the methods A, B-1, B-2 and Cmay be combined appropriately.

The starting compound (II) or its reactive derivative to be used in thisreaction can be easily obtained by the method disclosed in EP-A-334636or EP-A-438261, and the starting compound (III) can be easily obtainedaccording to the method disclosed in the literature reference [J. P.Greenstein and M. Winits, Chemistry of the Amino Acids Vols. I to III,John Wiley & Sons, Inc., New York.London (1961)].

The application of protective groups of each functional group to be usedupon necessity in the above respective production steps is known anddescribed in detail in the following literature references. [J. F. W.McOmine, Protective Groups in Organic Chemistry, Plenum Press, Londonand New York (1973); Pine, Hendrikson, Hammond, Organic Chemistry, 4thedition, [I]-[II], Hirokawa Shoten (1982); and M. Fieser and L. Fieser,Reagents for Organic Synthesis Vols. 1 to 13, Wiley-Interscience, NewYork, London, Sydney and Toronto (1969-1988)].

The amino group, hydroxyl group or mercapto group shown by X in thecompound [I] can be converted, upon necessity, into one another by aknown substituent-converting reaction on the pyrimidine ring [ProteinNucleic Acid Enzyme Extra Issue, Chemical Synthesis of Nucleic Acids,Kyoritsu Shuppan (1968)].

The compound (I) of this invention may form salts. Salts of basesinclude salts of alkali metals, alkaline earth metals, non-toxic metals,ammonium and quaternary ammonium, such as sodium, potassium, lithium,calcium, magnesium, aluminum, zinc, ammonium, trimethyl ammonium,triethanol ammonium, pyridinium and substituted pyridinium. Salts ofacids include salts of mineral acids such as hydrochloric acid, sulfuricacid, nitric acid, phosphoric acid and boric acid, and salts of organicacids such as oxalic acid, tartaric acid, acetic acid, trifluoroaceticacid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acidand camphorsulfonic acid.

The compound (I) or its salt produced by the above-mentioned method canbe isolated from the reaction mixture by conventional means forseparation, such as concentration, extraction with a solvent,chromatography and recrystallization.

The compounds (I) of this invention or their salts are storedefficiently in cells and have inhibitory action against one or morespecies of enzymes whose substrate is folic acid or its relatedcompounds. Therefore, these compounds can be used for the therapy of notonly papillary carcinoma, leukemia, mastocarcinoma, derencephaloepidermal cancer, squamous cell carcinoma, small cell cancer of the lungand lymphatic sarcoma, which have so far been treated with MTX, but alsoother various tumors, singly or in combination with any other antitumoragent.

The compounds (I) and pharmaceutically acceptable salts thereof, whenused as antitumor agents, can be administered to warm-blooded animals,particularly mammals (e.g. human, monkey, dog, cat, rabbit, rat, mice,etc.) orally and non-orally as they are or in the forms of, for example,powder, granules, tablets, capsules, suppositories and injections, whichare prepared according to conventional methods using pharmaceuticallyacceptable vehicles, excipients, and diluents. The dose varies accordingto the animals, diseases, symptoms, compounds and administration routes;for example, the daily dose is about 2.0 to 200 mg, preferably 1.0 to200 mg, more preferably 2.5 to 50 mg in terms of the compound (I) or itssalt of this invention per kg of body weight of a warm-blooded animaldescribed above for oral administration, and about 0.5 to 100 mg/kg,preferably 1.0 to 100 mg/kg, more preferably 1.0 to 2.0 mg/kg fornon-oral administration. Injections may be administered intramuscularly,intraperitoneally, subcutaneously or intravenously. By theseadministrations, therapy of tumors can be performed without significanttoxicity.

The preparations described above are produced by per se known processes.The above-mentioned oral preparations, for example, tablets may beproduced by suitable combination with a binder (e.g.hydroxypropylcellulose, hydroxypropylmethylcellulose, macrogol, etc.), adisintegrator (e.g. starch, calcium carboxylmethylcellulose, etc.) and alubricant (e.g. magnesium stearate, talc, etc.).

The non-oral preparations, for example, injections may be produced bysuitable combination with an isotonizing agent (e.g. glucose,D-sorbitol, D-mannitol, sodium chloride, etc.), an antiseptic (e.g.benzyl alcohol, chlorobutanol, methyl p-hydroxybenzoate, propylp-hydroxybenzoate, etc.) and a buffer (e.g. phosphate buffer, sodiumacetate buffer, etc.). A practical process of production of tabletscomprises, for example, mixing about 1.0 to 50 mg of the compound (I) ora salt thereof of this invention, 100 to 500 mg of lactose, about 50 to100 mg of corn starch and about 5 to 20 mg of hydroxypropylcellulose forpreparation of one tablet by a conventional means, granulating, mixingwith corn starch and magnesium stearate and tabletting, so that tabletseach weighing about 100 to 500 mg with a diameter of about 3 to 10 mmare obtained. The tablets may be coated with a mixture of acetone andethanol, the mixture containing hydroxypropylmethylcellulose phthalate(about 10 to 20 mg per tablet) and castor oil (0.5 to 2.0 mg per tablet)at a concentration of about 5 to 10%, to give enteric coated tablets. Asa practical example for the preparation of an injection, about 2.0 to 50mg of a sodium salt of the compound (I) of this invention forpreparation of one ampoule may 1 be dissolved in about 2 ml ofphysiological saline, sealing the resultant solution in an ampoule andsterilizing the ampoule at 110° C. for about 30 minutes or 2 bedissolved in a solution of about 10 to 40 mg of mannitol or sorbitol, inabout 2 ml of sterile distilled water, filling the solution into anampoule and freeze-drying and sealing the ampoule, so that an injectioncan be prepared. For use of the freeze-dried compound for subcutaneous,intravenous or intramuscular injection, the ampoule is opened and thecontent is dissolved in, for example, physiological saline so that theconcentration of the compound may be about 0.5 to 100 mg/ml, preferably1.0 to 50 mg/ml, more preferably 1.0 to 20 mg/ml.

Experiments showing pharmacological effects of the compounds (I) ortheir salts in the present invention are given below.

The thymidylate acid synthase (TS) inhibiting action, the5-aminoimidazole-4-carboxyamide ribonucleotide transformylase (AICARTF)inhibiting action and the Meth A fibrosarcoma cell growth inhibitingaction in vitro, of the typical subject compounds in the presentinvention obtained in the Working Examples described below, weredetermined by the following methods.

Experiment 1 Determination of TS inhibiting action

A roughly refined fraction of TS was prepared from Meth A fibrosarcomacells serially cultivated in vitro. For culturing the cells, Eagle'sminimum essential medium containing 10%(^(v) /v) bovine fetal serum[MEM; Nissui Pharmaceutical Co. Ltd.] was used. The cells in logarithmicgrowth were recovered and washed twice with a physiological aqueoussaline solution buffered with phosphate, which was then suspended in0.2M sucrose, 0.01M Tris.HCl buffer solution (pH 7.5). The cells weredestroyed by ultrasonic wave, and the suspension was subjected tocentrifugation (100,000×g), then the supernatant was collected. Usingbovine γ-groblin as the standard protein, the protein concentration wasmeasured by using a protein.dye reagent (Bio-Rad), so that the proteinconcentration was adjusted to 10 mg/ml.

Enzymic reaction was conducted by partially modifying the methoddescribed in Biochemistry 5, 3546 (1966). The composition of thereaction medium was as follows; 0.058%(^(v) /v) formaldehyde, 6.78 mMsodium fluoride, 0.2 mM 2-mercapto ethanol, 6.24 mg/ml bovine serumalbumin, 80 μM 2,-deoxyuridine-5'-1 phosphoric acid (dUMP), 80 μMtetrahydrofolic acid, 2 mg/ml roughly refined TS fraction and 0.173MTris.HCl buffer solution (pH 7.5). To the reaction medium were added thecompounds of various concentrations obtained in Working Examples. 540KBg of [³ H]dUMP relative to 50 μl of the ultimate reaction medium wasadded, and the reaction was allowed to proceed for 1 hour at 37° C. in a96-microwell plate. After completion of the reaction, 26.65%trichloroacetic acid and 3.33 mg/ml dUMP were added to suspend thereaction. To the reaction mixture was added 220 μl of 11.4 mg/mlactivated charcoal. The whole mixture was subjected to centrifugation,and the radioactivity in 100 μl of the supernatant was measured byliquid scintillation counter to determine the concentration required for50% inhibiting the TS activity (IC₅₀), as shown in Table 1.

Experiment 2 Measurement of AICARTF inhibiting action

AICARTF was prepared from CCRF-CEM human lymphoblast leukemia cells bypartially modifying the method described in Biochemistry 20 337 (1981),S. J. Benkovic, et al.

The enzymic reaction was conducted by adding the compounds of variousconcentrations in Working Examples to 0.95 ml of a solution consistingof a 32.5 μM Tris.HCl buffer solution (pH 7.4), 5 μM 2-mercaptoethanol,25 μM potassium chloride, 0.1 μM (-)-10-formyl tetrahydrofolic acid andAICARTF, and, before starting the reaction, 0.05 ml of 1 mM5-aminoimidazole-4-carboxylamide ribonucleotide. The UV spectrum wasmeasured at 298 nm for 15 minutes at the interval of 15 seconds toevaluate by the increase of absorbance due to the production oftetrahydrofolic acid to determine the concentration required for 50%inhibiting the activity of AICARTF (IC₅₀). The results are shown inTable 1.

                  TABLE 1                                                         ______________________________________                                                       IC.sub.50 (μM)                                              Compound         TS    AICARTF                                                ______________________________________                                        Ex. 6            36    9                                                      Ex. 7            40    6                                                      Ex. 8            21    0.6                                                    Ex. 9            20    0.4                                                    Ex. 10           20    0.2                                                    ______________________________________                                    

Experiment 3 Measurement of inhibiting action against growth of Meth Afibrosarcoma (Meth A) cells

Meth A cells (2×10⁴ /ml) prepared by a conventional method wereinoculated into each well of the 12-microwell plate (2.0 ml in a well),and subjected to standing culture at 37° C. under 5% CO₂. The compoundsobtained in Working Examples dissolved at appropriate concentrationswere diluted to 2-10 times stepwise with an MEM (Nissui PharmaceuticalCo., Ltd.) solution, and the resulting dilution were added to theculture medium. The medium was again subjected to standing culture at37° C. under 5% CO₂ for 72 hours. Then, the total number of cells ateach concentration was measured by Coulter counter (Coulter Electronic,FL in U.S.A.), and the average value of the cells in three wells wasexpressed by the number of cells per milliliter.

The concentrations of the respective compounds required for decreasingthe number of cells in the untreated control group by 50% was made IC₅₀of the respective compounds. The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Compound      IC.sub.50 (μM)                                               ______________________________________                                        Ex. 6         0.61                                                            Ex. 7         0.58                                                            ______________________________________                                    

EXAMPLES

The following Reference Examples and Working Examples will explain thepresent invention more concretely.

In the following reference and working examples, NMR spectrum wasmeasured by means of Gemini 200 (200 MHz)-spectrometer, and all the δvalues were shown by ppm. Symbols in the examples have the followingmeanings.

    ______________________________________                                        s               singlet                                                       d               doublet                                                       t               triplet                                                       ABq             AB type quartet                                               dd              double doublet                                                dt              double triplet                                                m               multiplet                                                     br.             broad                                                         J               coupling constant                                             sh              shoulder                                                      Hz              hertz                                                         CDCl.sub.3      dichloroform                                                  DMSO-d.sub.6    dimethyl-sulfoxide                                            D.sub.2 O       Deuterium Oxide (water-d.sub.2)                               room temperature                                                                              10-30° C.                                              %               % (.sup.w /w)                                                 ______________________________________                                    

Reference Example 1

Production of methyl [N-(tert-butoxycarbonyl)-O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate

N-(tert-butoxycarbonyl)-L-glutamic acid methyl dicyclohexylamine (10.04g) was dispersed in a mixture of ethyl acetate (110 ml) and an aqueoussolution of 2M sodium hydrogensulfate (44 ml). The dispersion wasvigorously shaken in a separating funnel to give a solution. The aqueouslayer was discarded, and the organic layer was washed with 2M sodiumhydrogensulfate and a saturated aqueous saline solution, and was driedover anhydrous magnesium sulfate. The solvent was distilled off underreduced pressure. The residue and ethyl γ-benzyl-L-glutamatehydrochloride (7.18 g) were dissolved in dry dimethyformamide (50 ml).To the solution was added at 0° C. a solution of diethylcyanophosphorate (4.07 g) in dry dimethylformamide (50 ml), and themixture was stirred for 15 minutes. To the mixture was added dropwise adry dimethylformamide solution (50 ml) of triethylamine (4.82 g), whichwas stirred for one hour at 0° C., then for 15 hours at roomtemperature. The reaction mixture was diluted with benzene (1000 ml) andethyl acetate (2000 ml), which was washed with 5% hydrochloric acid,water, a saturated aqueous saline solution, a saturated aqueous solutionof sodium hydrogencarbonate, water and a saturated aqueous salinesolution, successively, then dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure. The residue wasrecrystallized from chloroform/petroleum ether to afford theabove-titled compound (8.79 g). Specific rotation: [α] D²² -23.4 (c=1.0,MeOH)

¹ H-NMR(CDCl₃)δ : 1.44(9H,s), 1.82-2.33(6H,m), 2.40-2.58(2H,m),3.73(6H,s), 4.20-4.37(1H,m), 4.61(1H,dt,J=7.6, 1.2Hz), 5.21(2H,s),5.20-5.33(1H,m), 6.46-6.60(1H,m), 7.36(5H,s)

IR(KBr): 3350, 1740, 1680, 1640, 1520 cm⁻¹

REFERENCE EXAMPLE 2

Production of [N-(tert-butoxycarbonyl)-O¹-methyl-γ-L-glutamyl]-L-glutamic acid methyl dicyclohexylamine salt

To a methanol solution (60 ml) of the compound obtained in ReferenceExample 1 (3.88 g) was added 10% palladium carbon (390 mg; manufacturedby Engelhard Co., Ltd. in U.S.A.), and the mixture was stirred for 2.5hours under hydrogen atmosphere. The catalyst was filtered off by usingcelite, then the filtrate was subjected to distillation under reducedpressure. The residue was dissolved in ether (50 ml), and the solutionwas cooled to 0° C., and there was added, while stirring,dicyclohexylamine (1.72 ml). The stirring was continued for 10 minutes,then precipitating crystals were collected by filtration and washed withether to give the above-titled compound (3.97 g). Specific rotation:[α]D²⁰ -17.8 (c=1.0, MeOH) IR(KBr): 3300, 2940, 2850, 1750, 1725, 1710,1670, 1540cm⁻¹

REFERENCE EXAMPLE 3

Production of [O¹ -methyl-γ-L-glutamyl]-γ-benzyl-L-glutamic acid methylhydrochloride

To a dichloromethane (250 ml) solution of the compound of ReferenceExample 2 (3.88 g) was added trifluoroacetic acid (6.5 ml). The mixturewas stirred for 4 hours at room temperature, then the solvent wasdistilled off under reduced pressure. The residue was dissolved in amixture of water and dichloromethane (1:1, 100 ml). The solution wasneutralized with a saturated aqueous solution of sodiumhydrogenphosphate, then the organic layer was separated. The organiclayer was washed with a saturated aqueous saline solution and dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was dissolved in ether (50 ml). To thesolution was added a 0.4 mol. hydrochloric acid ether solution (30 ml).The resultant crystalline precipitate was collected by filtration anddried to give the above-titled compound (2.73 g).

Specific rotation: [α]D²⁰ +25.9 (c=1.0, MeOH) ¹ H-NMR(CDCl₃)δ :1.95-2.70(8H,m), 3.67(3H,s), 3.75(3H,s), 4.19-4.22(1H,m),4.43-4.58(1H,m), 5.09(2H,s), 7.73(5H,s), 7.64-7.79(1H,m)

REFERENCE EXAMPLE 4

Production of methyl [N-(tert-butoxycarbonyl)-O¹-methyl-γ-L-glutamyl]-[O¹ -methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate

In substantially the same manner as Reference Example 1, theabove-titled compound (1.61 g) was obtained from the compound ofReference Example 2 (1.75 g) and γ-benzyl-L-glutamic acid methylhydrochloride (975 mg).

Specific rotation [α]D²¹ +31.1 (c=1.0, MeOH) ¹ H-NMR(CDCl₃) :1.44(9H,s), 1.70-1.55(12H,m), 3.73(9H,s), 4.22-4.36(1H,m),4.50-4.65(2H,m), 5.12(2H,s), 5.29(1H,d,J=8.6Hz), 6.67(1H,d,J=7.8Hz),6.80(1H,d,J=8.4Hz), 7.35(5H,s) IR(KBr): 3310, 1740, 1680, 1645, 1535cm⁻¹

REFERENCE EXAMPLE 5

Production of methyl [N-(tert-butoxycarbonyl)-O¹-methyl-γ-L-glutamyl]-[O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate

In substantially the same manner as Reference Example 1, theabove-titled compound (1.87 g) was obtained from the compound ofReference Example 2 (1.75 g) and the compound of Reference Example 3(1.385 g).

Specific rotation: [α]D²¹ -32.0° (c=1.0, MeOH)

¹ H-NMR(CDCl₃)δ : 1.44(9H,s), 1.72-2.56(16H,m), 3.71(9H,s), 3.73(3H,s),4.22-4.40(1H,m), 4.53-4.72(3H,m), 5.11(2H,s), 5.36(1H,d,J=9.0Hz),6.51(1H,d,J=7.6Hz), 6.97(1H,d,J=7.6Hz), 7.34(1H,m), 7.35(5H,s) IR(KBr):3300, 1740, 1680, 1645, 1535cm⁻¹

REFERENCE EXAMPLE 6

Production of methyl [N-(tert-butoxycarbonyl)-O¹-methyl-γ-L-glutamyl]-L-glutamate

To a methanol solution (25 ml) of the compound of Reference Example 4(830 mg) was added 10% palladium-carbon (85 mg). The mixture was stirredfor two hours under hydrogen atmosphere, then the catalyst was filteredoff by using celite. The filtrate was subjected to distillation to leavethe above-titled compound (710 mg).

Thin-layer chromatography (Silica Gel 60 F₂₅₄, manufactured by E. MerckA.G., in U.S.A., developing solvent; chloroform:methanol=10:1): Rf=0.10

This product was not purified any more, but used in Reference Example 8.

REFERENCE EXAMPLE 7

Production of methyl [N-(tert-butoxycarbonyl)-O¹-methyl-γ-L-glutamyl]-O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-L-glutamate

In substantially the same manner as Reference Example 6, theabove-titled compound (891 mg) was obtained from the compound ofReference Example 5 (1.018 g).

This-layer chromatography (Silica Gel 60 F₂₅₄, manufactured by E. MerckA.G., in U.S.A., developing solvent; chloroform:methanol=10:1): Rf=0.08

This product was not purified any more, but used in Reference Example 9.

REFERENCE EXAMPLE 8

Methyl [N-(tert-butoxycarbonyl)-O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-[O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate

The compound of Reference Example 6 (429 mg) and the compound ofReference Example 3 (514 mg) were dissolved in dry dimethylformamide (7ml). The solution was cooled to 0° C., and there was added a drydimethylformamide solution (7 ml) of diethyl cyanophosphate (141 mg).The mixture was stirred for 15 minutes at 0° C., and there was thenadded dropwise a dry dimethylformamide solution (7 ml) of triethylamine(337 mg). The mixture was stirred for one hour at 0° C., then for 20hours at room temperature. The reaction mixture was diluted withbenzene/ethyl acetate (1/2; 600 ml), washed with 5% hydrochloric acid,water, a saturated aqueous saline solution, a saturated aqueous solutionof sodium hydrogencarbonate, water and a saturated aqueous salinesolution, successively, then dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure. The residue wasrecrystallized from chloroform/petroleum ether to give the above-titledcompound (584 mg).

Specific rotation: [α]D²¹ -36.6 (c=1.0, MeOH) ¹ H-NMR(CDCl₃)δ :1.46(9H,s), 1.60-2.60(20H,m), 3.67(3H,s), 3.69(6H,s), 3.70(3H,s),3.75(3H,s), 4.28-4.76(5H,m), 5.11(2H,s), 5.20-5.30(1H,m),6.20-6.30(1H,m), 7.05-7.15(1H,m), 7.34(5H,s), 7.30-7.40(1H,m),7.56-7.63(1H,m) IR(KBr): 3300, 1740, 1680, 1645, 1540 cm⁻¹

REFERENCE EXAMPLE 9

Production of methyl [N-(tert-butoxycarbonyl)-O¹-methyl-γ-L-glutamyl]-[O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl-[O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate

In substantially the same manner as Reference Example 8, theabove-titled compound (878 mg) was obtained from the compound ofReference Example 3 (776 mg).

Specific rotation: [α]D²¹ -39.7 (c=1.0, MeOH)

¹ H-NMR (CDCl₃)δ : 1.46(9H,s), 1.77-2.70(24H,m), 3.64(3H,s), 3.67(3H,s),3.68(3H,s), 3.70(3H,s), 3.72(3H,s), 3.76(3H,s), 4.37-4.80(6H,s),5.11(2H,s), 5.22(1H,d,J=9.4 Hz), 6.22(1H,d,J=9.4 Hz), 7.17(1H,d,J=9.6Hz), 7.35(5H,s), 7.43(1H,d,J=8.6 Hz), 7.55(1H,d,J=8.4 Hz),7.69(1H,d,J=7.6 Hz) IR(KBr) : 3300, 1740, 1650, 1540 cm⁻¹

REFERENCE EXAMPLE 10

Production of [O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamic acid methyl trifluoroacetate

To a dichloromethane (10 ml) solution of the compound of ReferenceExample 4 (230 mg) was added trifluoroacetic acid (0.6 ml). The mixturewas stirred for 3 hours at room temperature. Then, the solvent wasdistilled off under reduced pressure to give the above-titled compound(298 mg).

This product was not purified any more, but used for the production ofcompounds in Working Examples.

REFERENCE EXAMPLE 11

Production of [O¹ -methyl-γ-L-glutamyl]-[O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamic acid methyl trifluoroacetate

In substantially the same procedure as Reference Example 10, theabove-titled compound (302 mg) was obtained from the compound ofReference Example 5.

This product was not purified any more, but used for the production ofcompounds in Working Examples.

REFERENCE EXAMPLE 12

Production of[O¹ -methyl-γ-L-glutamyl]-[O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-[O¹ -methyl-γ-L-glutamyl]-γ-benzyl-L-glutamic acidmethyl trifluoroacetate

In substantially the same procedure as Reference Example 10, theabove-titled compound (140 mg, 100%) was obtained from the compound ofReference Example 8.

This product was not purified any more, but used for the production ofcompounds in Working Examples.

REFERENCE EXAMPLE 13

Production of [O¹ -methyl-γ-L-glutamyl]-[O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-[O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamic acid methyl trifluroacetate

In substantially the same procedure as Reference Example 10, the abovetitled compound (562 mg, 100%) was obtained fro the compound ofReference Example 9.

This product was not purified any more, but used for the production ofcompound in Working Examples.

WORKING EXAMPLE 1

Production of methyl [N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate

[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoic acid(225 mg) and the compound of Reference Example 3 (343 mg) were dissolvedin dry dimethylformamide (5 ml). The solution was cooled to 0° C., andthere was added a dry dimethylformamide solution (5 ml) of diethylcyanophosphate (130 mg). The mixture was stirred for 15 minutes at 0°C., and there was then added dropwise a dry dimethylformamide solution(5 ml) of triethylamine (161 mg). The mixture was stirred for one hourat 0° C., then for 20 hours at room temperature. The solvent wasdistilled off under reduced pressure, and the residue was purified bymeans of a silica gel column chromatography [carrier;20 g, developingsolvent: chloroform:10% ammonia-containing ethanol=9:1] to give theabove-titled compound (292 g).

IR(KBr): 3350, 1730, 1600 cm⁻¹

¹ H-NMR(CDCl₃)δ : 1.90-2.39(14H,m), 3.67(3H,s), 3.76(3H,s), 4.59(2H,m),4.52-4.80(2H,m), 4.94(2H,m), 5.11(2H,s), 6.47(1H,m), 6.98(1H,d,J=8.0Hz), 7.20-7.36(7H,m), 7.53(1H,d,J=8.0 Hz), 7.75(2H,d,J=8.2 Hz),8.29(1H,m)

WORKING EXAMPLE 2

Production of methyl [N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate

By substantially the same procedure as Working Example 1, theabove-titled compound (159 mg) was obtained from4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl benzoic acid(120 mg) and the compound of Reference Example 10 (297 mg).

IR(KBr): 3360, 1740, 1650, 1600 cm⁻¹ ¹ H-NMR(CDCl₃)δ : 1.50-1.71(4H,m),1.82-2.00(4H,m), 2.02-2.25(2H,m), 2.30-2.43(6H,m), 2.50-2.63(2H,m),3.61(6H,s), 3.69(3H,s), 4.50(2H,m), 4.43-4.76(3H,m), 5.02(2H,s),5.17(2H,m), 6.41(1H,s), 7.11-7.40(10H,m), 7.70(2H,d,J=8.0 Hz),8.58(1H,m)

WORKING EXAMPLE 3

Production of methyl [N-[4-(3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-[O¹ -methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate

By substantially the same procedure as Working Example 1, theabove-titled compound (207 mg) was obtained from4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoic acid(84 mg) and the compound of Reference Example 11 (244 mg).

IR(KBr): 3370, 1740, 1650, 1600 cm⁻¹ ¹ H-NMR(CDCl₃)δ : 1.60-3.00(22H,m),3.63(3H,m), 3.70(6H,m), 3.74(3H,s), 4.42(2H,m), 4.52-4.87(4H,m),5.12(2H,s), 5.76(2H,s), 6.52(1H,s), 7.20-7.37(9H,m), 7.55(1H,d,J=8.0Hz), 7.80(2H,d,J=8.2 Hz), 7.85(1H,d,J=8.0 Hz), 9.35(1H,m)

WORKING EXAMPLE 4

Production of methyl [N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-[O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate

By substantially the same procedure as Working Example 1, theabove-titled compound (360 mg) was obtained from4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoic acid(162 mg) and the compound of Reference Example 12 (575 mg).

IR(KBr) 3400, 1740, 1650, 1610 cm⁻¹

¹ H-NMR(CDCl₃)δ : 1.58-2.97(26H,m), 3.64(3H,s), 3.67(3H,s), 3.69(3H,s),3.71(3H,s), 3.78(3H,s), 4.24(2H,m), 4.51-4.90(5H,m), 5.10(2H,m),5.90-6.14(2H,m), 6.55(1H,m), 7.17(1H,d,J=8.0 Hz), 7.22-7.42(8H,m),7.61(1H,m,J=8.0 Hz), 7.64(1H,d,J=8.0 Hz), 7.76(1H,d,J=8.0 Hz),7.82(2H,d,J=8.0 Hz), 9.53(1H,m)

WORKING EXAMPLE 5

Production of methyl [N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-O¹ -methyl-γ-L-glutamyl]-[O¹-methyl-γ-L-glutamyl]-[O¹ -methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate

By substantially the same procedure as Working Example 1, theabove-titled compound (271 mg) was obtained from4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoic acid(121 mg) and the compound of Reference Example 13 (503 mg).

IR(KBr): 3370, 1740, 1660, 1600 cm⁻¹

¹ H-NMR(CDCl₃)δ : 1.60-2.10(6H,m), 2.24-2.90(24H,m), 3.62(3H,s),3.67(3H,m), 3.68(3H,s), 3.69(3H,s), 3.73(3H,m), 3.79(3H,m), 4.25(2H,m),4.55-4.76(6H,m), 5.11(2H,s), 5.85(2H,m), 6.54(1H,m), 7.13(1H,d,J=8.0Hz), 7.20-7.38(8H,m), 7.50-7.70(4H,m), 7.81(2H,d,J=8.2 Hz), 9.32(1H,m)

WORKING EXAMPLE 6

Production of[N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-.gamma.-L-glutamyl]-L-glutamicacid

The compound of Working Example 1 (281 mg) was dissolved in a mixture ofwater (12 ml) and tetrahydrofuran (8 ml). To the solution was added 1Nsodium hydroxide (1.84 ml), and the mixture was stirred for 4 hours atroom temperature. Tetrahydrofuran was distilled off under reducedpressure. A small amount of impurities was filtered off with a milliporefilter, then the filtrate was neutralized with 1N hydrochloric acid(1.84 ml), and was left standing for a few minutes. Water was removedwith a pipette. To the residue was added ether/methanol, then the wallof the vessel was rubbed with a spatula to cause formation of whitepowder. The powder was collected by filtration and dried to obtain theabove-titled compound (157 mg).

IR(KBr): 3340, 3200, 2930, 1730, 1640 cm⁻¹

¹ H-NMR(DMSO-d₆)δ : 1.64-2.12(6H,m),,2.26-2.32(4H,m), 2.58-2.71(4H,m),4.06-4.17(1H,m), 4.22-4.40(1H,m), 5.56(2H,m), 6.16(2H,m), 6.45(1H,s),7.30(2H,d,J=8.2 Hz), 7.80(2H,d,J=8.2 Hz), 8.12(1H,d,J=8.0 Hz),8.58(1H,d,J=8.0 Hz), 10.52(1H,m)

WORKING EXAMPLE 7

Production of[N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-.gamma.-L-glutamyl]-γ-L-glutamyl-L-glutamicacid

By substantially the same procedure as Working Example 6, theabove-titled compound (187 mg) was obtained from the compound of WorkingExample 2 (296 mg).

IR(KBr): 3340, 3200, 2930, 1720, 1640 cm⁻¹

¹ H-NMR(DMSO-d₆)δ 1.68-2.00(6H,m), 2.07-2.32(8H,m), 2.62-2.77(4H,m),4.10-4.21(2H,m), 4.23-4.42(1H,m), 5.60-5.73(2H,m), 6.31(2H,m),6.47(1H,s), 7.30(2H,d,J=8.2 Hz), 7.81(2H,d,J=8.2 Hz), 8.05(1H,d,J=8.0Hz), 8.13(1H,d,J=8.0 Hz), 8.59(1H,d,J=8.0 Hz), 10.57(1H,m)

WORKING EXAMPLE 8

Production of[N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-.gamma.-L-glutamyl]-γ-L-glutamyl-γ-L-glutamicacid

By substantially the same procedure as Working Example 6, theabove-titled compound (137 mg) was obtained form the compound of WorkingExample 3 (199 mg).

IR(KBr): 3340, 1730, 1650 cm⁻¹

¹ H-NMR(DMSO-d₆)δ : 1.65-1.73(18H,m), 2.60-2.78(4H,m), 4.13-4.20(3H,m),4.25-4.45(1H,m), 5.90-6.10(2H,m), 6.54(1H,m), 6.74(2H,m),7.30(2H,d,J=8.4 Hz), 7.82(2H,d,J=8.4 Hz), 8.05(1H,d,J=8.0 Hz),8.10(1H,d,J=8.0 Hz), 8.17(1H,d,J=8.0 Hz), 8.60(1H,d,J=8.0 Hz),10.78(1H,m)

WORKING EXAMPLE 9

Production of[N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-.gamma.-L-glutamyl]-γ-L-glutamyl-γ-L-glutamyl-γ-L-glutamyl-L-glutamicacid

By substantially the same procedure as Working Example 6, theabove-titled compound (127 mg) was obtained from the compound of WorkingExample 4 (206 mg).

IR(KBr): 3340, 1730, 1650 cm⁻¹

¹ H-NMR(DMSO-d₆)δ : 1.55-2.34(22H,m), 2.60-2.80(4H,m), 4.10-4.21(4H,m),4.25-4.43(1H,m), 5.90-6.10(2H,m), 6.54(1H,s), 6.78(2H,m),7.30(2H,d,J=8.4 Hz), 7.84(2H,d,J=8.4 Hz), 8.03-8.26(4H,m),8.61(1H,d,J=8.0 Hz), 10.82(1H,m)

WORKING EXAMPLE 10

Production of[N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-.gamma.-L-glutamyl]-γ-L-glutamyl-γ-L-glutamyl-γ-L-glutamyl-.gamma.-L-glutamyl-L-glutamicacid

By substantially the same procedure as Working Example 6, theabove-titled compound (60 mg) was obtained from the compound of WorkingExample 5 (126 mg).

IR(KBr): 3340, 1730, 1650 cm⁻¹

¹ H-NMR(DMSO-d₆)δ : 1.60-2.40(26H,m), 2.61-2.75(4H,m), 4.07-4.23(5H,m),4.30-4.40(1H,m), 5.90-6.15(2H,m), 6.54(1H,s), 6.78(2H,m),7.30(2H,d,J=8.2 Hz), 7.82(2H,d,J=8.2 Hz), 8.00-8.16(5H,m),8.60(1H,d,J=8.0 Hz), 10.82(1H,m)

WORKING EXAMPLE 11

Production of[N-[4-[2-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethylthio]benzoyl]-γ-L-glutamyl]-L-glutamicacid

By substantially the same procedure as Working Example 1, methyl[N-[4-[2-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethylthio]benzoyl]-O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate was synthesized from4-[2-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethylthio]benzoicacid (331 mg) and the compound of Reference Example 3 (517 mg). Thewhole amount of this product was subjected to the same hydrolysis as inWorking Example 6 to afford the above-titled compound (294 mg) as amixture of diastereoisomer.

¹ H-NMR(Me₂ SO-d₆)δ : 1.62-1.80(2H,m), 1.82-2.20(6H,m), 2.21-2.37(2H,m),2.90-3.03(2H,m), 3.22-3.37(2H,m), 3.45-3.62(1H,m), 4.03-4.18(1H,m),4.23-4.37(1H,m), 6.23(1H, bs), 6.31(1H, bs), 6.46(1H, bs), 6.55(1H, bs),6.90(1H,s), 7.31(1H, d,J=9 Hz), 7.33(1H, d,J=9 Hz), 7.77(1H, d,J=9 Hz),7.79(1H, d,J=9 Hz), 8.10(0.5H,d,J=8 Hz), 8.17(0.5H,d, J=8 Hz),8.63(0.5H,d,J=8 Hz), 8.69(0.5H,d,J=8 Hz), 10.60(1H,s)

WORKING EXAMPLE 12

Production of [N-[4-[3-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-γ-L-glutamyl]-L-glutamic acid

By substantially the same procedure as Working Example 1, methylN-[4-[3-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-O¹-methyl-γ-L-benzyl-L-glutamate was synthesized from4-[3-(2-amino-4-hydroxy-7H- pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoicacid (313 mg) and the compound of Reference Example 3 (517 mg). Thewhole amount of this product was subjected to the same hydrolysis as inWorking Example 6 to afford the above-titled compound (282 mg).

¹ H-NMR(DMSO-d₆)δ : 1.78-2.17(6H,m), 2.23-2.41(4H,m), 2.53-2.80(4H,m),4.01-4.12(1H,m), 4.30-4.47(1H,m), 5.92(2H,s), 6.36(1H,s), 7.28(2H,d,J=8Hz), 7.79(2H,d,J=8 Hz), 8.12(1H,d,J=7.8 Hz), 8.55(1H,d,J=7.8 Hz),10.10(1H,s)

WORKING EXAMPLE 13

Production ofN-[4-[N-[2-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid

By substantially the same procedure as Working Example 1, methylN-[4-[N-[2-(2,4-diamino-6,7-dihydro5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]benzoyl-O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate was synthesized from4-[N-[2-(2,4-diamino-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]benzoicacid (329 mg) and the compound of Reference Example 3 (517 mg). Thewhole amount of this product was subjected to the same hydrolysis as inWorking Example 6 to afford the above-titled compound (276 mg).

¹ H-NMR(DMSO-d₆ +D₂ O)δ : 1.41-1.65(1H,m), 1.75-2.11(5H,m),2.28(4H,t,J=7 Hz), 2.93(3H,s), 3.13-3.45(4H,m), 3.52-3.66(1H,m),3.99-4.12(1H,m), 4.20-4.34(1H,m), 6.72(2H,dd,J=9 Hz, 2 Hz),7.71(2H,d,J=9 Hz)

WORKING EXAMPLE 14

Production of[N-[5-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]-2-thenoyl]-.gamma.-L-glutamyl]-L-glutamicacid

By substantially the same procedure as Working Example 1, methyl[N-[5-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]-2-thenoyl]-O¹-methyl-γ-L-glutamyl]-γ-benzyl-L-glutamate was synthesized from5-[3-(2,4-diamino-7H- pyrrolo[2,3-d]pyrimidin-5-yl)propyl]-2-thiophenecarboxylic acid trifluoroacetic acid salt (431 mg) and the compound ofReference Example 3 (517 mg). The whole amount of this product wassubjected to the same hydrolysis as in Working Example 6 to afford theabove-titled compound (305 mg).

¹ H-NMR(DMSO-d₆)δ : 1.76-2.18(6H,m), 2.27-2.40(4H,m), 2.70(2H,t,J=7.6Hz), 2.85(2H,t,J=7.6 Hz), 4.02-4.14(1H,m), 4.24-4.37(1H,m), 5.60(2H,s),6.21(2H,s), 6.47(1H,s), 6.87(1H,d,J=3.6 Hz), 7.68(1H,d,J=3.6 Hz),8.10(1H,d,J=7.6 Hz), 8.51(1H,d,J=7.6 Hz), 10.52(1H,s)

WORKING EXAMPLE 15

[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid

By substantially the same procedure as Working Example 1 and 6, theabove-titled compound was obtained.

¹ H-NMR(Me₂ SO-d₆)δ : 1.76-2.15(4H,m), 2.20-2 33(4H,m), 2.78-3.03(4H,m),4.12-4.22(1H,m), 4.28-4.41(1H,m), 6.01(2H,s), 6.32(1H,d,J=2.0Hz),7.28(2H,d,J=8.0Hz), 7.78(2H,d,J=8.0Hz), 8.06(1H,d,J=8.0Hz),8.52(1H,d,J=8.0Hz), 10.16(1H,s), 10.60(1H,s)

WORKING EXAMPLE 16

[N-[4-[N-2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid

By substantially the same procedure as Working Example 1 and 6, theabove-titled compound was obtained.

¹ H-NMR(Me₂ SO-d₆ +D₂ O)δ : 1.79-2.14 (4H,m), 2.21-2.35(4H,m),2.85(2H,t,J=7.0Hz), 3.31(2H,t,J=7.0Hz), 4.10-4.22(1H,m),4.31-4.43(1H,m), 6.50(1H,s), 6.63(2H,d,J=8.8Hz), 7.65(2H,d,J=8.8Hz)

WORKING EXAMPLE 17

[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid

By substantially the same procedure as Working Example 1 and 6, theabove-titled compound was obtained.

¹ H-NMR(Me₂ SO-d₆ +D₂ O)δ : 1.77-2.15(4H,m), 2.20-2.35(4H,m),2.77(2H,t,J=7.4Hz), 2.98(3H,s), 3.66(2H,t,J=7.4Hz), 4.09-4.20(1H,m),4.31-4.45(1H,m), 6.43(1H,d,J=1.8Hz), 6.88(2H,d,J=8.8Hz),7.76(2H,d,J=8.8Hz)

WORKING EXAMPLE 18

[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]benzoyl]-γ-L-glutamyl]-L-glutamicacid

By substantially the same procedure as Working Example 1 and 6, theabove-titled compound was obtained.

¹ H-NMR(Me₂ SO-d₆ +D₂ O)δ : 1.78-2.15(4H,m), 2.20-2.36(4H,m),2.86(2H,m), 3.12(1H,s), 3.66(2H,m), 4.10-4.21(1H,m), 4.19(2H,s),4.32-4.46(1H,m), 6.47(1H,d,J=2.0Hz), 6.99(2H,d,J=9.0Hz),7.77(2H,d,J=9.0Hz)

WORKING EXAMPLE 19

[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid

By substantially the same procedure as Working Example 1 and 6, theabove-titled compound was obtained.

¹ H-NMR(Me₂ SO-d₆ +D₂ O)δ : 1.80-2.16(4H,m), 2.18-2.33(4H,m),2.79(2H,m), 3.05(3H,s), 3.64(2H,m), 4.07-4.19(1H,m), 4.33-4.22(1H,m),5.94(1H,d,J=4.4Hz), 6.49(1H,d,J=2.0Hz), 7.56(1H,d,J=4.4Hz)

WORKING EXAMPLE 20

[N-[5-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl-N-propargylamino]-2-thenoyl]-γ-L-glutamyl]-L-glutamicacid

By substantially the same procedure as Working Example 1 and 6, theabove-titled compound was obtained.

¹ H-NMR(Me₂ SO-d₆ +D₂ O)δ : 1.79-2.14(4H,m), 2.19-2.34(4H,m),2.85(2H,m), 3.10(1H,s), 3.65(2H,m), 4.11-4.23(1H,m), 4.18(2H,s),4.34-4.45(1H,m), 6.05(1H,d,J=4.4Hz), 6.50(1H,d,J=2.0Hz),7.58(2H,d,J=4.4Hz)

WORKING EXAMPLE 21

[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-methylamino]-2-fluorobenzoyl]-γ-L-glutamyl]-L-glutamicacid

By substantially the same procedure as Working Example 1 and 6, theabove-titled compound was obtained.

¹ H-NMR(Me₂ SO-d₆ +D₂ O)δ : 1.75-2.17(4H,m), 2.22-2.36(4H,m),2.76(2H,m), 2.99(3H,s), 3.64(2H,m), 4.05-4.18(1H,m), 4.29-4.43(1H,m),6.40(1H,dd,J=2.2,14.8Hz), 6.48(1H,s), 6.50(1H,dd,J=2.2,8.6Hz),7.75(1H,t,8.6Hz)

WORKING EXAMPLE 22

[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]-2-fluorobenzoyl]-γ-L-glutamyl]-L-glutamicacid

By substantially the same procedure as Working Example 1 and 6, theabove-titled compound was obtained.

¹ H-NMR(Me₂ SO-d₆ +D₂ O)δ : 2.87(2H,m), 3.09(1H,s), 3.66(2H,m),4.04-4.19(1H,m), 4.20(2H,s), 4.33-4.43(1H,m), 6.42(1H,dd,J=2.2,14.8Hz),6.47(1H,s), 6.51(1H,dd,J=2.2,8.6Hz), 7.74(1H,t,J=8.6Hz)

WORKING EXAMPLE 23

[N-[4-[N-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-N-propargylamino]-2-chlorobenzoyl]-γ-L-glutamyl]-L-glutamicacid

By substantially the same procedure as Working Example 1 and 6, theabove-titled compound was obtained.

¹ H-NMR(Me₂ SO-d₆ +D₂ O)δ : 1.79-2.20(4H,m), 2.23-2.38(4H,m),2.84(2H,m), 3.15(1H,s), 3.65(2H,m), 4.19(2H,s), 4.06-4.20(1H,m),4.27-4.43(1H,m), 6.46(1H,s), 7.00(1H,d,J=8.8Hz), 7.01(1H,s),7.33(1H,d,J=8.8Hz)

What is claimed is:
 1. A compound of the formula ##STR12## wherein aring A stands for an optionally hydrogenated pyrrole ring, X stands foran amino group, hydroxyl group or mercapto group, R stands for hydrogenor a lower alkyl group, m denotes an integer of 2 to 4, R may bedifferent in each of m repeating units, COOR¹ and COOR² independentlystand for a carboxyl group which may be esterified with (1) a C₁₋₅ alkylgroup, (2) a benzyl group which may be substituted with a nitro or aC₁₋₄ alkoxy group, or (3) a phenyl group which may be substituted with anitro or a C₁₋₄ alkoxy group, n denotes an integer of 2 to 6 and R¹ maybe different in each of the n repeating units, or a pharmaceuticallyacceptable salt thereof.
 2. A compound as claimed in claim 1, wherein Xstands for amino group.
 3. A compound as claimed in claim 1, wherein Xstands for hydroxyl group.
 4. A compound as claimed in claim 1, whereinCOOR¹ and COOR² independently stand for a carboxyl group which may beesterified with(1) a C₁₋₅ alkyl group, or (2) a benzyl group.
 5. Acompound as claimed in claim 1, wherein n denotes an integer of 2 or 3.6. A compound as claimed in claim 1, wherein COOR¹ and COOR² both standfor COOH.
 7. Compound of claim 1 which is[N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-.gamma.-L-glutamyl]-L-glutamicacid.
 8. Compound of claim 1 which is[N-[4-[2-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-.gamma.-L-glutamyl]-L-glutamicacid.
 9. Compound of claim 1 which is[N-[4-[2-(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-γ-L-glutamyl]-L-glutamicacid.
 10. An anti-tumor composition which comprises an effective amountof a compound as claimed in claim 1 or a pharmaceutically acceptablesalt thereof, together with a pharmaceutically acceptable carrier orcarriers.
 11. An agent for inhibiting thymidylic acid synthase or5-aminoimidazole-4-carboxyamide ribonucleotide transformylase or growthof Meth A fibrosarcoma cell which comprises an effective amount of acompound as claimed in claim 1 or a pharmaceutically acceptable saltthereof.
 12. A method for treating tumors selected from the groupconsisting of papillary carcinoma, leukemia, mastocarcinoma,derencephalo epidermal cancer, squamous cell carcinoma, small cellcancer of the lung and lymphatic sarcoma which comprises administeringan effective amount of a compound as claimed in claim 1 or itspharmaceutically acceptable salt together with a pharmaceuticallyacceptable carrier or diluent to a mammal.